Parkinson?s Disease (PD) afflicts ~1% of humans and prevalence increases with age. Initial symptoms include tremors in extremities, but PD progresses over years, affecting additional limbs, with impairment of mood/behavior/cognition. Familial PD makes up ~10% of cases while certain chemicals/environmental toxicants (e.g., rotenone, paraquat, maneb) increase the risk of PD and much evidence points to genetic x environmental interactions. The overall goal of our project is to develop an assay system, utilizing dopaminergic neurons derived from induced pluripotent stem cells (iPSC-DNs and iPSC-MGs) and automated digital microscopy and associated methods (Kinetic Image Cytometry [KIC] and High Content Analysis [HCA]) to enable screening of environmental toxicants for potential PD toxicity. In phase I we demonstrated that iPSC-DN featuring the A53T-?-Syn mutation (which causes early-onset PD, with high penetrance) have increased spontaneous activity (calcium and voltage transients) and increased sensitivity to PD-linked toxicants vs. neurons with wt-?-Syn, which demonstrates the feasibility of our approach. In Phase II we will collaborate with researchers at The Parkinson?s Institute and Clinical Center, to further develop our assay methods, utilizing iPSC-DNs that feature G2019S-LRRK2, a common, PD-associated mutation, that has very recently been linked to increased susceptibility to environmental toxicants, iPSC lines representing both sexes, and lines derived from subjects with sporadic PD. We will also develop a coculture system with both iPSC-DNs and iPSC-MGs in the culture wells. The resulting assay system will enable the most comprehensive screening of environmental toxicants for potential PD-inducing effects, yet available. Potential customers for the assay include government agencies, such as the US EPA and the National Toxicology Program, and global agencies concerned with the effects of industrial/agricultural chemicals on human health.